Our previous work has shown that a) adenosine induces sinus slowing and atrioventricular (AV) block in a number of animal models and recently in humans, and, b) adenosine may be involved in the ischemia and/or hypoxia induced sinus bradycardia and AV block. The principal objectives of this proposal are to define and characterize the cellular mechanism(s) underlying the actions of adenosine on sinoatrial (SA) and AV nodes as well as the working atrial myocardium. The experiments will be conducted on small SA node preparations, 200-300 Mum, which preclude pacemaker shift, and on single isolated atrial and AV node cells. The isolated cells and small SA node are ideal preparations for the proposed voltage clamp studies. In addition, the studies will allow precise determination of the kinetics of the adenosine response. We will use the two microelectrode voltage clamp technique for ionic current analysis, ion sensitive electrodes for the direct measurement of potassium efflux and pressure-injection (adenosine applied extracellularly) for the kinetic analysis of the adenosine response. We plan to use these techniques to characterize the actions of adenosine, adenosine analogs, antagonists, and adenosine uptake inhibitors on all three cell types. In addition to providing knowledge concerning the cellular mechanism and kinetics of the response in SA node, AV node and atrial cells to adenosine, these studies will provide new perspectives in the clinical evaluation of impulse formation and conduction disturbances. Through our multi-leveled approach, studying single isolated cells to humans, we have gained some insight into the potential significance of adenosine as a modulator of cardiac function. Therefore, the present proposal will allow us to extend our efforts in using this type of approach towards understanding the mechanism of action of adenosine in the heart.